DESCRIPTION: (taken from the application). Anesthetics are the most toxic of drugs administered by physicians, and yet we have little idea how they work. Improvement in the drugs can only occur with an enhanced understanding of their binding sites, and the atomic interactions responsible for binding. In this subproject, we intend to localize and characterize the important features of inhaled anesthetic binding sites. We hypothesize that protein internal cavities are essential for specific binding of anesthetics, and that their volume controls the effect the drugs will have on protein stability and dynamics. We also hypothesize that deep hydrophobic pockets are important, and that pocket/cavity polarity improves anesthetic binding affinity. We will test these hypotheses by graphically analyzing a subset of about 100 protein structures deposited with the Protein Data Bank for cavity and pocket features, and then correlating a subset of these with binding and stabilization using photolabeling fluorescence quenching and amide hydrogen exchange. Those proteins showing specific binding will be co-crystallized with the anesthetic to verify location of binding, and the atomic interactions responsible. These experiments with biological proteins build on our experience and success with peptide models. In collaboration with Project 2, designed cavities will be created and modified in both water soluble and membrane inserted helical peptide bundles, and then studied with photolabeling and amide hydrogen exchange to monitor the binding event Crystallization studies of these bundles for high resolution structural information will be conducted. Finally, many proteins exist for which no high-resolution structural detail is known (especially the large membrane proteins responsible for much CNS signaling), but photolabeling can assign location in the primary structure, and yield an idea of binding parameters. Halothane photolabeling, while useful, has limitations that dictate the development of novel photolabels based on the diazo- or diazirine group. In collaboration with Dr. William Dailey of the Department of Chemistry, we will design, synthesize and characterize new compounds to mimic an alkane, an ether, and a non-immobilizer compound to use on these complex proteins in the future. These studies will establish or refute the importance of pre-existing cavities or pockets in anesthetic binding, and thus establish the generality of important anesthetic-binding features. Further this work will provide a foundation for a unitary hypothesis of anesthetic-induced protein dysfunction: anesthetics stabilize the protein conformer with optimal cavity features, reducing flexibility and thereby hindering the shifts in conformational equilibria that underlie activity.